Summary Accurate DNA replication is essential for the maintenance of the genetic information during cell proliferation in all organisms. Defects in DNA replication factors are associated with human diseases including cancer. The complexity of eukaryotic DNA replication has previously confounded efforts to directly address biochemical mechanisms involved in this process in vitro. We have developed functional in vitro assays that allow us to overcome this limitation. We have shown previously that pre-replicative complexes (pre-RCs), which license origins in G1 phase for subsequent activation in S phase, can be reconstituted with proteins purified from budding yeast, Saccharomyces cerevisiae. Importantly, we recently found that reconstituted pre-RCs also support regulated DNA replication in vitro, exhibiting the fundamental hallmarks of cellular DNA replication. These functional assays enable us to use interdisciplinary approaches to address key problems in eukaryotic DNA replication initiation that were previously intractable, forming the basis for experiments proposed here: Aim 1) Mcm2-7 is loaded onto origins in inactive form by the pre-RC in G1 phase. Activation of the Mcm2-7 helicase, and concomitant replisome assembly in S phase, requires the formation of a pre-initiation complex (pre-IC) around the pre-RC. The goal is to delineate the mechanism of origin activation by the pre-IC using reconstitution studies with purified budding yeast proteins. We will characterize the order of pre-IC assembly, determine the structure of pre-IC subassemblies, define the steps that induce local origin unwinding, and assign biochemical functions to pre-IC components. These studies form the basis for our long-term goal to reconstitute the entire eukaryotic DNA replication reaction in vitro. Aim 2) Mcm2-7 complexes can passively slide along DNA prior to activation of the helicase. We will characterize this sliding ability and test its role in initiatin site determination, which has important implications for the eukaryotic origin specification mechanism. Upon activation, Mcm2-7 complexes actively translocate along DNA in a directed manner to unwind DNA at the fork. We will use biochemistry, electron microscopy, and advanced single molecule fluorescence microscopy to analyze the structural configuration of active Mcm2-7 helicase complexes and determine if Mcm2-7 hexamers of opposite orientation physically separate or remain associated during elongation. These studies will provide insight into the Mcm2-7 DNA unwinding mechanism, which underlies eukaryotic replisome organization. Aim 3) We have found previously that two Mcm2-7 hexamers are cooperatively loaded in opposite orientation around double stranded DNA by the pre-RC. To elucidate the mechanism for Mcm2-7 loading we will define the interaction network of Cdc6 during pre-RC assembly using site-specific protein-protein cross-linking in vitro and in vivo. This approach will both identify the functional interaction partners for Cdc6 in the pre-RC and inform structural models for the pre-RC.